Abstract：
OBJECTIVE: Liver fibrosis remains a complication derived from a chronic Hepatitis C Virus (HCV) infection even when it is resolved, and no liver antifibrotic drug has been approved. Molecular mechanisms on hepatocytes and activation of hepatic stellate cells (HSCs) play a central role in liver fibrogenesis. To elucidate molecular mechanisms, it is important to analyze pathway regulation during HSC activation and HCV infection.
METHODS: We evaluate the fibrosis-associated molecular mechanisms during a co-culture of human HSCs (LX2), with human hepatocytes (Huh7) that express HCV NS5A or Core protein. We evaluated LX2 activation induced by HCV NS5A or Core expression in Huh7 cells during co-culture. We determined a fibrosis-associated gene expression profile in Huh7 that expresses NS5A or Core proteins during the co-culture with LX2.
RESULTS: We observed that NS5A induced 8.3-, 6.7- and 4-fold changes and that Core induced 6.5-, 1.8-, and 6.2-fold changes in the collagen1, TGFβ1, and timp1 gene expression, respectively, in LX2 co-cultured with transfected Huh7. In addition, NS5A induced the expression of 30 genes while Core induced 41 genes and reduced the expression of 30 genes related to fibrosis in Huh7 cells during the co-culture with LX2, compared to control. The molecular pathways enriched from the gene expression profile were involved in TGFB signaling and the organization of extracellular matrix.
CONCLUSIONS: We demonstrated that HCV NS5A and Core protein expression regulate LX2 activation. NS5A-induced LX2 activation, in turn, regulates diverse fibrosis-related gene expression at different levels in Huh7, which can be further analyzed as potential antifibrotic targets during HCV infection.
METHODS: We evaluate the fibrosis-associated molecular mechanisms during a co-culture of human HSCs (LX2), with human hepatocytes (Huh7) that express HCV NS5A or Core protein. We evaluated LX2 activation induced by HCV NS5A or Core expression in Huh7 cells during co-culture. We determined a fibrosis-associated gene expression profile in Huh7 that expresses NS5A or Core proteins during the co-culture with LX2.
RESULTS: We observed that NS5A induced 8.3-, 6.7- and 4-fold changes and that Core induced 6.5-, 1.8-, and 6.2-fold changes in the collagen1, TGFβ1, and timp1 gene expression, respectively, in LX2 co-cultured with transfected Huh7. In addition, NS5A induced the expression of 30 genes while Core induced 41 genes and reduced the expression of 30 genes related to fibrosis in Huh7 cells during the co-culture with LX2, compared to control. The molecular pathways enriched from the gene expression profile were involved in TGFB signaling and the organization of extracellular matrix.
CONCLUSIONS: We demonstrated that HCV NS5A and Core protein expression regulate LX2 activation. NS5A-induced LX2 activation, in turn, regulates diverse fibrosis-related gene expression at different levels in Huh7, which can be further analyzed as potential antifibrotic targets during HCV infection.
摘要：
目的：肝纤维化仍然是由慢性丙型肝炎病毒（HCV）感染引起的并发症，即使它已经解决，并且还没有批准肝脏抗纤维化药物。肝细胞的分子机制和肝星状细胞（HSC）的激活在肝纤维化中发挥重要作用。为了阐明分子机制，分析HSC激活和HCV感染过程中的通路调控非常重要。
方法：我们评估了人类HSC（LX2）共培养过程中纤维化相关的分子机制，与表达HCVNS5A或核心蛋白的人肝细胞(Huh7)。我们评估了在共培养期间由HCVNS5A或核心表达在Huh7细胞中诱导的LX2活化。我们确定了在与LX2共培养期间表达NS5A或Core蛋白的Huh7中的纤维化相关基因表达谱。
结果：我们观察到NS5A诱导8.3-，6.7倍和4倍变化，核心诱导6.5-，1.8-,胶原蛋白1，TGFβ1和timp1基因表达的6.2倍变化，分别,在与转染的Huh7共培养的LX2中。此外,与对照相比,在与LX2共培养期间,NS5A诱导了30个基因的表达,而Core诱导了41个基因,并降低了Huh7细胞中与纤维化相关的30个基因的表达。从基因表达谱中富集的分子途径涉及TGFB信号传导和细胞外基质的组织。
结论：我们证明了HCVNS5A和Core蛋白表达调节LX2活化。NS5A诱导的LX2激活，反过来,在Huh7中以不同的水平调节不同的纤维化相关基因表达，这可以作为HCV感染期间潜在的抗纤维化靶标进一步分析。
方法：我们评估了人类HSC（LX2）共培养过程中纤维化相关的分子机制，与表达HCVNS5A或核心蛋白的人肝细胞(Huh7)。我们评估了在共培养期间由HCVNS5A或核心表达在Huh7细胞中诱导的LX2活化。我们确定了在与LX2共培养期间表达NS5A或Core蛋白的Huh7中的纤维化相关基因表达谱。
结果：我们观察到NS5A诱导8.3-，6.7倍和4倍变化，核心诱导6.5-，1.8-,胶原蛋白1，TGFβ1和timp1基因表达的6.2倍变化，分别,在与转染的Huh7共培养的LX2中。此外,与对照相比,在与LX2共培养期间,NS5A诱导了30个基因的表达,而Core诱导了41个基因,并降低了Huh7细胞中与纤维化相关的30个基因的表达。从基因表达谱中富集的分子途径涉及TGFB信号传导和细胞外基质的组织。
结论：我们证明了HCVNS5A和Core蛋白表达调节LX2活化。NS5A诱导的LX2激活，反过来,在Huh7中以不同的水平调节不同的纤维化相关基因表达，这可以作为HCV感染期间潜在的抗纤维化靶标进一步分析。
